Donor apoptotic cell–based therapy for effective inhibition of donor‐specific memory T and B cells to promote long‐term allograft survival in allosensitized recipients

The mechanisms that regulate B cell memory and the rapid recall response to antigen remain poorly defined. This study focuses on the rapid expression of B cell memory upon antigen recall in vivo, and the replenishment of quiescent B cell memory that follows. Based on expression of CD138 and B220, we reveal a unique and major subtype of antigen-specific memory B cells (B220−CD138−) that are distinct from antibody-secreting B cells (B220+/−CD138+) and B220+CD138− memory B cells. These nonsecreting somatically mutated B220− memory responders rapidly dominate the splenic response and comprise >95% of antigen-specific memory B cells that migrate to the bone marrow. By day 42 after recall, the predominant quiescent memory B cell population in the spleen (75–85%) and the bone marrow (>95%) expresses the B220− phenotype. Upon adoptive transfer, B220− memory B cells proliferate to a lesser degree but produce greater amounts of antibody than their B220+ counterparts. The pattern of cellular differentiation after transfer indicates that B220− memory B cells act as stable self-replenishing intermediates that arise from B220+ memory B cells and produce antibody-secreting cells on rechallenge with antigen. Cell surface phenotype and Ig isotype expression divide the B220− compartment into two main subsets with distinct patterns of integrin and coreceptor expression. Thus, we identify new cellular components of B cell memory and propose a model for long-term protective immunity that is regulated by a complex balance of committed memory B cells with subspecialized immune function.

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Comparable Long-Term Persistence of Anti-FVIII Antibodies in Hemophilic E17 Mice on Different Genetic Backgrounds Despite Significant Differences in the Amplitude of the Immune Responses.

Blood ◽

10.1182/blood.v108.11.1027.1027 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1027-1027

Author(s):

Natalie Bauer ◽

Christina Hausl ◽

Rafi U. Ahmad ◽

Bernhard Baumgartner ◽

Hans Peter Schwarz ◽

...

Keyword(s):

Immune Response ◽

B Cells ◽

Genetic Background ◽

Neutralizing Antibodies ◽

Plasma Cells ◽

Memory B Cells ◽

Mouse Strains ◽

Igg Antibodies ◽

Specific Memory

Abstract About 30% of patients with severe hemophilia A develop neutralizing antibodies against FVIII (FVIII inhibitors) following replacement therapy. The type of FVIII gene mutation as well as other predisposing genetic factors contribute to the inhibitor phenotype. Based on these findings, we asked if the genetic background modulates the long-term persistence of anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells in the E17 murine hemophilia model. Furthermore, we asked if the recently described inhibition of memory-B-cell re-stimulation by high doses of FVIII is influenced by the genetic background of the murine model. E17 mice on two different genetic backgrounds (C57Bl/6J and Balb/c) were treated with four doses of 200 ng human FVIII at weekly intervals. Anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells were followed up to 12 months after the last dose of FVIII. Antibody titers and subclasses of antibodies (IgM, IgG1, IgG2a, IgG2b, IgG3) were measured by ELISA. Antibody secreting plasma cells in spleen and bone marrow were detected by ELISPOT as described (Hausl et al., Thromb Haemost 2002). The re-stimulation of FVIII-specific memory B cells was studied as described recently (Hausl et al., Blood 2005). Anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells were first detectable in E17 Balb/c mice. IgM antibodies in the circulation and IgM secreting plasma cells in the spleen were observed after the first dose of FVIII, IgG antibodies and IgG secreting plasma cells after the second dose. No anti-FVIII antibodies after the first dose of FVIII were observed in E17 C57BL/6J mice but both IgM and IgG antibodies as well as IgM and IgG producing plasma cells were detectable after the second dose of FVIII. The antibody response involved all IgG subclasses in both mouse strains. However, IgG1 was dominant in E17 Balb/c mice whereas IgG2a was dominant in E17 C57BL/6J mice. When the in vitro restimulation of FVIII-specific memory B cells was examined, similar patterns were observed for both mouse strains. Low concentrations of FVIII between 10 and 100 ng/ml FVIII restimulated memory B cells and induced their differentiation into antibody secreting plasma cells whereas high concentrations of FVIII between 1,000 and 20,000 ng/ml FVIII inhibited memory-B-cell-restimulation. These results indicate that the dose-dependent effect of FVIII on the restimulation of FVIII-specific memory B cells does not depend on the genetic background. The major difference between both hemophilic mouse strains was the amplitude of the anti-FVIII immune response. Peak titers of anti-FVIII antibodies and peak concentrations of anti-FVIII antibody secreting plasma cells in spleen and bone marrow were significantly higher in E17 C57BL/6J mice than in E17 Balb/c mice. Whether or not higher ELISA titers correlate with higher Bethesda titers of neutralizing antibodies is currently being investigated. Despite the substantial differences in the amplitude of the immune response, anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells persisted for the whole observation period of 12 months after the last dose of FVIII in both mouse strains. We conclude that the amplitude of the anti-FVIII immune response in hemophilic mice is significantly different between E17 C57BL/6J and E17 Balb/c mice. However, the persistence of the immune response is comparable.

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Double-Unit Umbilical Cord Blood Transplantation Induces MHC-Matched Single-Unit Dominance with Development of Immunocompetent Lymphocytes in Recipient Mice

Blood ◽

10.1182/blood.v120.21.4097.4097 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4097-4097

Author(s):

Koichi Ito ◽

Akira Nakano ◽

Kyoko Ito ◽

Ikuo Kashiwakura ◽

Hideaki Sato

Keyword(s):

Bone Marrow ◽

B Cells ◽

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Single Unit ◽

T And B Cells ◽

X Ray ◽

Cytotoxic Cells

Abstract Abstract 4097 Background: Double-unit umbilical cord blood cell (dUCBC) transplantation has emerged as an effective strategy for improving the engraftment of umbilical cord blood stem cells in the bone marrow of recipients. Due to a lack convenient animal models, analyses of the differentiation capacity of dUCBC in recipients have been limited to in vivo xenogeneic experiments and clinical observations. In the present study, we evaluated the characteristics of immune reconstitution induced by dUCBC transplantation in mice. Materials and Methods: Natural killer cells were depleted from female C57BL/6 (B6) [H-2b] recipient mice by intraperitoneal administration of rabbit anti-asialo GM1 polyclonal antibody 1 day before transplantation. On the following day, the lethal X-ray-irradiated B6 recipients were given transplants of three different combinations of dUCBC {group (1) GFP-Tg B6 [H-2b] and BALB/c [H-2d]; group (2) GFP-Tg B6 [H-2b] and C3H [H-2k]; group (3) BALB/c [H-2d] and C3H [H-2k]}, each combination containing an equal number of cells. At 16 weeks after transplantation, reconstitution of immune cells was evaluated by flow cytometric analysis utilizing specific antibodies against lineage markers such as CD3 (T cells), CD45R/B220 (B cells), CD11b (macrophages), or Ly-6G (granulocytes). The donor origin of each lineage population was determined by anti-H-2Kk (for C3H) and/or H-2Kd (for BALB/c) antibody staining. GFP+ lineage cells were identified as being of B6 donor origin. Skin grafting was then performed in all recipients to assess the functional maturity of the newly developed T and B cells induced by dUCBC transplantation. Results: The survival rate at 16 weeks after transplantation was 73% (8/11) for case (1), 92% (12/13) for case (2), and 50% (3/6) for case (3). In the great majority of cases (1) and (2), in which dUCBC were administered as a stem cell source, the MHC-matched single unit from GFP-Tg B6 acts as the sole source of long-term hematopoiesis (75% (6/8) for case (1); 100% (12/12) for case (2)). CD3+ T cell peripheral blood chimerism from BALB/c was observed in two of the eight B6 survival recipient mice in case (1) at an early stage of hematopoiesis, predicting the long-term engrafting unit. On the other hand, hematopoiesis in case (3) with fully allogenenic dUCBC transplantation was reconstituted by the B6 recipients' own X-ray-resistant hematopoietic stem cells (HSC). Our results indicate that MHC-matched UCBC-HSC predominantly engraft in the recipient's bone marrow after dUCBC transplantation. However, the nature of this selective mechanism remains largely unknown. In all cases, alloreactive cytotoxic cells in recipient may participate in such selection. In dUCBC transplantation, the included allogeneic cells probably act as stimulators for promoting the differentiation and maturation of MHC-matched HSC through activation of certain types of signal transduction (for example, through cytokine secretion). Currently, we are investigating the possible presence of alloreactive cytotoxic cells in bone marrow. Functionally, these recipients were tolerant of skin grafted from B6, whereas they rejected skin from BALB/c and C3H within 20 days, indicating that both CD4+ helper and CD8+ killer T cells were functionally mature in the recipient mice. Correspondingly, only the alloantibody to BALB/c and C3H was produced in the recipients. One of two chimeric recipient mice in case (1) reacted to only C3H skin with T and B cells. Conclusions: dUCBC transplantation clearly rescued mice that had been subjected to lethal X-ray irradiation. Furthermore, our observations indicate that T and B cells derived from dUCBCs transplants are immunologically fully competent with the ability to distinguish self from non-self MHC antigens. However, a clear understanding of the mechanisms underlying the predominant engraftment of MHC-matched HSCs in the recipient's bone marrow will be necessary. Disclosures: No relevant conflicts of interest to declare.

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Detection of SARS-CoV-2 Specific Memory B cells to Delineate Long-Term COVID-19 Immunity

10.22541/au.161074580.02596064/v1 ◽

2021 ◽

Author(s):

Constantin Thieme ◽

Mohamed Abou-el-Enein ◽

Enrico Fritsche ◽

Moritz Anft ◽

Sarah Skrzypczyk ◽

...

Keyword(s):

B Cells ◽

Vaccine Development ◽

Health Concern ◽

Public Health Response ◽

Specific Memory ◽

Healthy Donors ◽

Health Response ◽

Standard Flow Cytometry ◽

Novel Coronavirus

Background: The ongoing COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, represents a serious worldwide health concern. A deeper understanding of the immune response to SARS-CoV-2 will be required to refine vaccine development and efficacy as well as to evaluate long-term immunity in convalescent patients. With this in mind, we investigated the formation of SARS-CoV-2 specific BMEMORY cells from patient blood samples. Methods: A standard flow cytometry-based protocol for the detection of SARS-CoV-2 specific B cells was applied using fluorochrome-coupled SARS-CoV-2 spike (S) full-length protein. Cohorts of 26 central European convalescent mild/moderate COVID-19 patients and 14 healthy donors were assessed for the levels of SARS-CoV-2 S- specific BMEMORY cells. Results: Overall B cell composition was not affected by SARS-CoV-2 infection in convalescent patients. Our analysis of SARS-CoV-2 specific BMEMORY cells in samples collected at different time points revealed that S-protein specific B cells remain in peripheral blood at least up to 6 months after COVID-19 diagnosis. Conclusions: Detection of SARS-CoV-2 specific BMEMORY cells may improve our understanding of the long-term adaptive immunity in response to SARS-CoV-2, allowing for an improved public health response and vaccine development during the COVID-19 pandemic. Further validation of the study in larger and more diverse populations and a more extended observation period will be required.

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Fibrinogen-Like Protein 2/Fibroleukin Induces Long-Term Allograft Survival in a Rat Model through Regulatory B Cells

PLoS ONE ◽

10.1371/journal.pone.0119686 ◽

2015 ◽

Vol 10 (3) ◽

pp. e0119686 ◽

Cited By ~ 17

Author(s):

Séverine Bézie ◽

Elodie Picarda ◽

Laurent Tesson ◽

Karine Renaudin ◽

Justine Durand ◽

...

Keyword(s):

B Cells ◽

Rat Model ◽

Regulatory B Cells ◽

Allograft Survival

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Selection of Criteria for Assessing Protective Immunity at Different Times of Experimental Tularemia in a Mouse Model

Biotekhnologiya ◽

10.21519/0234-2758-2021-37-4-65-77 ◽

2021 ◽

Vol 37 (4) ◽

pp. 65-77

Author(s):

G.M. Titareva ◽

A.N. Mokrievich ◽

T.I. Kombarova ◽

G.M. Vakhrameeva ◽

R.I. Mironova ◽

...

Keyword(s):

Immune Response ◽

B Cells ◽

Vaccine Strain ◽

Protective Immunity ◽

T And B Cells ◽

Immunological Parameters ◽

Protective Properties ◽

Virulent Strains ◽

Ifn Γ

It is known that the body's defense against infection by the intracellular bacterium Francisella tularensis is provided by the activation of the cellular and humoral immune response. However, their role in long-term protection (25 years and more) against virulent strains of F. tularensis is not well understood. The identification of clear criteria for assessing protective immunity to the tularemia causative agent at different times after vaccination will make it possible to more efficiently develop new genetically determined vaccine strains. The goal of our research was to select and assess immunological parameters reflecting the protective properties of the vaccine strain F. tularensis 15 NIIEG and its derivatives, F. tularensis 15/23-1∆recA and F. tularensis 15/ 23-1/sodB∆recA, in the long term after immunization. To assess the functional activity of T and B cells, flow cytometry was used.The assessment of the production of cytokines IFN-γ, IL-4, IL-10, IL-17A and titers of specific class G immunoglobulins to F. tularensis lipopolysaccharide (LPS)in blood serum was performed by ELISA on days 30, 60, 90 and 180 after immunization. Evaluation of the protective properties of vaccine preparations in the above-mentioned terms was carried out after subcutaneous infection with test-infecting virulent strains, Schu and 503 of tularensis and holarctica subspecies, respectively. It was shown that vaccination with the studied strains in 100% of cases protected from infection with the strain 503 of the holarctica subspecies, analogous to the vaccine strain. When infected with a virulent Schu strain of the hetrologous tularensis subspecies, a decrease in the effectiveness of protection was observed starting from 60 days after immunization. Evaluation of immunological parameters showed that at all studied periods after immunization, IgG antibodies to F. tularensis LPS were detected in the blood sera of immunized mice. In vitro experiments on stimulation of immune response in spleen lymphocytes of vaccinated mice to the F. tularensis antigen showed a significant increase in the level of secreted IFN-γ, a slight increase in secreted IL-10 and an enhanced expression of the CD69 molecule on the surface of T and B cells. Thus, the level of IFN-γ and the expression of the CD69 molecule on the surface of T and B cells in response to restimulation of lymphocytes of immune animals with tularemia antigen can serve as criteria for immune protection in experimental tularemia in a mouse model at different times after vaccination. Key words: vaccine strain, Fransicella tularensis, immunogenicity, protection, memory T cells, IgG, cellular immunity Funding - The work was supported by the Branch Program of the Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing.

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Evidence of SARS-Cov-2-specific memory B cells six months after vaccination with BNT162b2 mRNA vaccine

10.1101/2021.07.12.21259864 ◽

2021 ◽

Author(s):

Annalisa Ciabattini ◽

Gabiria Pastore ◽

Fabio Fiorino ◽

Jacopo Polvere ◽

Simone Lucchesi ◽

...

Keyword(s):

B Cells ◽

Memory B Cells ◽

Limited Information ◽

Specific Memory ◽

Cell Immunity ◽

Antibody Titers ◽

Specific Igg ◽

Humoral Responses

SARS-CoV-2 mRNA vaccines have demonstrated high efficacy and immunogenicity, but limited information is currently available on memory B cells generation and long-term persistence. Here, we investigated Spike-specific memory B cells and humoral responses in 145 subjects, up to six months after the BNT162b2 vaccine (Comirnaty) administration. Spike-specific antibody titers peaked 7 days after the second dose and significant titers and neutralizing activity were still observed after six months, despite a progressive decline over time. Concomitant to antibody reduction, Spike-specific memory B cells, mostly IgG class-switched, increased in blood of vaccinees and persisted six months after vaccination. Following in vitro restimulation, circulating memory B cells reactivated and produced Spike-specific antibodies. A high frequency of Spike-specific IgG+ plasmablasts, identified by computational analysis 7 days after boost, positively correlated with the generation of IgG+ memory B cells at six months. These data demonstrate that mRNA BNT162b2 vaccine elicits strong B cell immunity with Spike-specific memory B cells that still persist six months after vaccination, playing a crucial role for rapid response to SARS-CoV-2 virus encounter.

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Effects of Immunosuppressive Drugs on Hematopoietic Engraftment after Simultaneous Infusion of Apoptotic Cells and Bone Marrow Cells.

Blood ◽

10.1182/blood.v106.11.5207.5207 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5207-5207

Author(s):

Emeline Masson ◽

François Kleinclauss ◽

Sylvain Perruche ◽

Amandine Radlovic ◽

Hugues Bittard ◽

...

Keyword(s):

Bone Marrow ◽

Skin Graft ◽

Apoptotic Cell ◽

Tolerance Induction ◽

Favorable Effect ◽

Apoptotic Cells ◽

Skin Grafts ◽

Chimeric Mice ◽

Cell Based Therapy

Abstract We have recently showed in a murine bone marrow transplantation (BMT) model that apoptotic cell infusion simultaneously with a bone marrow (BM) graft favors hematopoietic engraftment, and induces a long term tolerance restricted to BM cell allo-antigens. This effect is observed whatever the origin of apoptotic cells and does not lead to the development of autoimmunity or GvHD. This favorable effect on engraftment was obtained without immunosuppressive (IS) drugs. In human BMT, IS treatment is always required. The aim of this study was to investigate the interactions between apoptotic cells and IS drugs, especially since some of them might interfere with other strategies of tolerance induction. The roles of ciclosporin A (CSA), mycophenolate mofetil (MMF), sirolimus (Rapa) and LF150195 (LF), a deoxyspergualin analog, were tested. Recipient Balb/c mice (H2d) were sublethally irradiated (6 Gy) 1 day before receiving 106 BM cells with or without apoptotic cells (5x106 γ-irradiated splenocytes) from donor FvB mice (H2q). Drugs were injected IP: CSA 50mg/kg/d from day 0 (d0) to d14, MMF 20mg/kg/d from d0 to d14, Rapa 1.5mg/kg/d from d0 to d14, and LF 0.625mg/kg/d from d0 to d10. Engraftment was assessed by flow cytometry analysis in peripheral blood. Skin grafts from BM donor mice were performed >70 days after BMT, to assess the influence of IS drugs on our tolerance induction protocol. CSA treatment didn’t favor engraftment in mice receiving BM alone (22% engraftment vs 20% without IS drug, p=NS), and induced only transient chimerism (<d70). CSA inhibited the effect of apoptotic cells on engraftment (13% engraftment with BM, apoptotic cells and CSA, vs 41% with BM and apoptotic cells without IS drug, p<0.05). MMF didn’t promote engraftment in mice receiving BM alone (11% engraftment vs 20%, p=NS), and didn’t have any effect when associated with apoptotic cell infusion (30% engraftment vs 41%, p=NS). Rapa was very effective in permitting engraftment of BM alone (93% engraftment vs 20%, p<0.05), and when associated with apoptotic cells, Rapa achieved a stable and long term chimerism in all grafted mice. LF increased engraftment after infusion of BM cells alone (40% engraftment vs 20%, p<0.05), and after BM and apoptotic cell infusion, 93 % of mice engrafted (p<0.05). Engraftment was stable in all mice (up to 600 days post-BMT), except for the group receiving CSA and BM alone. Skin grafts were tolerated in all chimeric mice (up to 400 days after skin graft), and were rejected in the same way by non chimeric mice (either because there wasn’t any BM engraftment, or because chimerism was lost at the time of skin graft) and control naive mice. This study confirms that IS drugs can interact with tolerance induction protocols, and particularly with our model of cell-based therapy using IV apoptotic cell infusion to promote BM engraftment. CSA shouldn’t be associated with apoptotic cells, as it seems to antagonize the favoring effect on engraftment. MMF could be used, since it doesn’t hamper the action of apoptotic cells. Rapa is highly effective even without apoptotic cells, and doesn’t interfere with their action. LF seems to be the most interesting drug in our model, because its effects are synergic with apoptotic cells in promoting engraftment. In clinical settings, Rapa and MMF can be used in addition to apoptotic cell infusion to favor engraftment after reduced intensity conditioning regimens.

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